The present invention relates generally to plant molecular biology and genetic approaches for engineering enhanced and broad-spectrum resistance to bacterial diseases in plants.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Bacterial blight of rice (Oryza sativa) caused by Xanthomonas oryzae pv. oryzae is one of the most destructive bacterial diseases throughout the world (Mew, 1987). X. oryzae pv. oryzae enters susceptible cultivars via hydathodes, multiplies in the epithelium, and moves to the xylem vessels to cause systemic infection (Ronald, 1997). In resistant cultivars, reduced growth of the avirulent race is reflected in lower bacterial populations, reduced lesion development, activation of defense gene expression and changes of the cell wall and plasma membrane that are adjacent to avirulent bacterial cells (Young et al., 1996; Gu et al., 2005).
Bacterial leaf streak, another bacterial disease of rice caused by Xanthomonas oryzae pv. oryzicola, is an emerging problem in rice growing countries (Nino-Liu et al. 2006). X. oryzae pv. oryzicola penetrates the leaf mainly through stomata, multiplies in the substomatal cavity and then colonizes the intercellular spaces of the parenchyma (Nino-Liu et al. 2006). Like X. oryzae pv. oryzae, X. oryzae pv. oryzicola may also gain access through wounds, but it remains restricted to the apoplast of the mesophyll tissue and does not invade the xylem (Ou, 1985). X. oryzae pv. oryzicola exudes from natural openings in the leaf in chains or strands, or under moist conditions as small beads of ooze, which is a typical sign of bacterial leaf streak (Nino-Liu et al. 2006).
The use of resistant cultivars is the most economical and effective method to control bacterial blight disease (Ogawa, 1993). Race-specific interaction between rice and X. oryzae pv. oryzae is thought to follow the classic gene-for-gene concept (Flor, 1971). The products of plant resistance (R) gene recognize or interact with an elicitor molecule presumably encoded by an avirulence (Avr) gene from the pathogen, leading to the activation of a cascade of defense responses and effectively inhibit pathogen invasion. Currently, more than two dozen R genes or loci against X. oryzae pv. oryzae have been identified in rice, most of them providing complete, race-specific resistance (Kinoshita, 1995; Lin et al., 1996; Zhang et al., 1998; Khush and Angeles, 1999; Gao et al., 2001; Chen et al., 2002; Yang et al., 2003; He et al., 2006). Four dominant R genes, Xa21 (Song et al., 1995), Xa1 (Yoshimura et al., 1998), Xa26 (Sun et al., 2004) and Xa27 (Gu et al., 2005), and two recessive R genes, xa5 (Iyer and McCouch, 2004) and xa13 (Chu et al., 2006), have been isolated by map-based cloning.
Few studies have been conducted on control methods for bacterial leaf streak. As is the case for bacterial blight, in practice, host genetic resistance is the most important control measure for bacterial leaf streak, although it is so far limited to quantitative resistance (Gnanamanickam et al., 1999; Sheng et al., 2005; Tang et al., 2000).
Unlike other R genes isolated from dicots, the R genes for bacterial blight resistance isolated from rice encode products with great diversity. Xa21 and Xa26 encode receptor-like proteins (Song et al., 1995; Sun et al., 2004). Biochemical analysis of the putative kinase domain of Xa21 revealed that Xa21 encodes a functional serine threonine protein kinase capable of autophosphorylation on multiple sites (Liu et al., 2002). The Xa1 gene product contains nucleotide binding sites (NBS) and leucine-rich repeats (LRR) and is a member of the largest class of plant R proteins (Yoshimura et al., 1998). The xa5 gene encodes the gamma, subunit of transcriptional factor IIA (TFIIA), a eukaryotic transcriptional factor with no previously known role in disease resistance (Iyer and McCouch, 2004). The xa13 gene encodes an MtN3-like protein (Chu et al., 2006). The dominant allele of the gene presumably functions in both disease susceptibility and pollen development (Chu et al., 2006). The recently isolated Xa27 gene encodes a novel protein that has no apparent sequence homology to proteins from organisms other than rice (Gu et al., 2005).
Currently, five Avr genes have been isolated from X. oryzae pv. oryzae and all of them belong to the AvrBs3 family of type-III effectors. Four type-III effectors AvrXa3 (Li et al., 2004; Lee et al., 2005), AvrXa7 (Hopkins et al., 1992; Vera Cruz et al., 2000), AvrXa10 (Hopkins et al., 1992; Zhu et al., 1998) and avrXa27 (Gu et al., 2005) are recognized respectively by four cognate dominant R genes, Xa3, Xa7, Xa10 and Xa27, in the host. Given that Xa5 genotypes are susceptible to X. oryzae pv. oryzae, it is speculated that type-III effector Avrxa5 targets wild-type Xa5 for pathogenicity. The mutated protein xa5 cannot be targeted by Avrxa5 during pathogenesis, so the plant containing homozygous xa5 alleles were resistant or “non-susceptible” to X. oryzae pv. oryzae infection (Schornack et al., 2006).
Xa27 and avrXa27 are the first pair of R and Avr genes isolated from rice and X. oryzae pv. oryzae, respectively (Gu et al., 2005). avrXa27 is a member of the AvrBs3/PthA family of nuclear localized type-III effectors with 16.5 thirty-four amino acid direct repeats in the central repetitive domain and a conserved C-terminal region containing three nuclear localization signal (NLS) motifs and a transcription activation domain (AD). The central repetitive region determines the avrXa27-elicited resistance specificity while the NLS motifs and AD domain are required for Xa27-dependent elicitation and resistance. Unexpectedly, the resistant and susceptible parental lines of the Xa27 mapping population encode identical Xa27 proteins. The polymorphism of nucleotide sequences between the presumed Xa27/xa27 promoters raised the possibility that the two alleles differ in their expression. Indeed, only the Xa27 allele, but not the xa27 allele, was detected by Northern blot analysis. Further studies revealed that expression of the Xa27 allele occurs only when a rice plant is challenged by bacteria harboring avrXa27, but not the mutated isogenic strains lacking avrXa27. These data suggests that the resistance specificity of Xa27 towards incompatible pathogens involves the differential expression of the Xa27 allele in the presence of the avrXa27 effector.
The Avr gene has bifunctional signals in virulence and host recognition (Kjemtrup et al., 2000; Alfano et al., 2004; Yang et al., 2000). When an Avr gene performs its virulent function, it suppresses host defenses during pathogenesis in compatible interactions. However, when the Avr gene acts as an avirulent gene, it betrays the pathogen to plant defense by being recognized by the cognate host R gene and triggering hypersensitive response (HR) in incompatible interactions. The virulent function of several Avr proteins expressed in planta was found to cause suppression of host defenses, cell death or necrosis in plants lacking cognate R genes (Gopalan et al., 1996; McNellis et al., 1998; Duan et al., 1998; Chen et al., 2000; Chen et al., 2004; Hauck et al., 2003). In plants carrying specific R genes, the Avr proteins expressed in planta can elicit an HR or cause lethality (Gopalan et al., 1996; Scofield et al., 1996; Tang et al., 1996; Van den Ackerveken et al., 1996; de Feyter et al., 1998; McNellis et al., 1998; Stevens et al., 1998).
There is a need to develop methods of generating disease resistance in plants and in particular to methods of generating broad-spectrum resistance to bacterial blight and enhanced resistance to bacterial leaf streak.